Cooperation of ERK and SCFSkp2 for MKP-1 destruction provides a positive feedback regulation of proliferating signaling

Abstract

The dual-specificity MAPK phosphatase MKP-1/CL100/DUSP1 is an inducible nuclear protein controlled by p44/42 MAPK (ERK1/2) in a negative feedback mechanism to inhibit kinase activity. Here, we report on the molecular basis for a novel positive feedback mechanism to sustain ERK activation by triggering MKP-1 proteolysis. Active ERK2 docking to the DEF motif (FXFP, residues 339-342) of N-terminally truncated MKP-1 in vitro initiated phosphorylation at the Ser296/Ser323 domain, which was not affected by substituting Ala for Ser at Ser359/Ser364. The DEF and Ser296/Ser323 sites were essential for ubiquitin-mediated MKP-1 proteolysis stimulated by MKK1-ERK signaling in H293 cells, whereas the N-terminal domain and Ser359/Ser364 sites were dispensable. ERK activation by serum increased the endogenous level of ubiquitinated phospho-Ser296 MKP-1 and the degradation of MKP-1. Intriguingly, active ERK-promoted phospho-Ser296 MKP-1 bound to SCFSkp2 ubiquitin ligase in vivo and in vitro. Forced expression of Skp2 enhanced MKP-1 polyubiquitination and proteolysis upon ERK activation, whereas depletion of endogenous Skp2 suppressed such events. The kinetics of ERK signaling stimulated by serum correlated with the endogenous MKP-1 degradation rate in a Skp2-dependent manner. Thus, MKP-1 proteolysis can be achieved via ERK and SCFSkp2 cooperation, thereby sustaining ERK activation. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.